1. Field of the Invention
This invention relates generally to a process of extracting procyanidins by alkaline hydrolysis, and more particularly to a process of extracting procyanidins from procyanidin-containing plant materials, such as cranberry pomace, using alkaline hydrolysis followed by acidification in order to obtain procyanidins in a stable form for use in various applications.
2. Description of the Related Art
Procyanidins are a class of polyphenolic compounds that impart astringency and bitterness to many plant products. In plants, procyanidins are believed to serve as a defense mechanism against potential predators because their bitterness and astringency is undesirable to animals, insects and microbes. (Aron, P. M., et al. “Flavan-3-ols: Nature, occurrence and biological activity.” Mol. Nutr. Food Res. 2008, 52, 79-104). Procyanidins are formed via the condensation of the flavan-3-ols catechin and epicatechin and comprise two (2) to several monomeric units. Structurally, the monomeric units may be linked in one of three ways. The “B”-type linkage is the most common and consists of 4β→8 linkage between units. Units connected by both a 2β→O-7 and a 4β→8 are more rigid than ‘B’-type linkages and are denoted as “A”-type. The final type of linkage is the “C”-type linkage, which consists of a C-4→C-6 linkage. (Id.). The ability of cranberries to prevent urinary tract infections has been attributed to the presence of procyanidins containing ‘A’-type linkages. (Foo, L. Y., et al. “The structure of cranberry proanthocyanidins which inhibit adherence of uropathogenic p-fimbriated Escherichia coli in vitro.” Phytochem. 2000, 54, 173-181). The bioavailability of procyanidins is dependent upon the size of the molecule, with monomers and dimers being relatively easily absorbed and those larger than trimers being typically poorly absorbed. (Donovan, J. L., et al. “Procyanidins are not bioavailable in rats fed a single meal containing a grape seed extract or the procyanidin B3.” Br. J. Nutr. 2002, 87, 299-306; and Holt, R. R., et al. “Procyanidin dimer B2 [epicatechin-(4b-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa.” Am. J. Clin. Nutr. 2002, 76, 798-804).
Polyphenolic compounds, including procyanidins, are commonly perceived to be found mainly in the vacuoles of plants where they are separated from other cellular components. However, many may also be associated with other cellular components, such as the cell wall, especially after cell injury when vacuoles may rupture. This results in the release of phenolic compounds, which may then associate with other cellular components, such as cell wall polysaccharides through hydrogen bonding and hydrophobic interactions. (Pinelo, M., et al. “Upgrading of grape skins: Significance of plant cell-wall structural components and extraction techniques for phenol release.” Trends Food Sci. Tech. 2006, 17, 579-590). Procyanidins in particular have a strong affinity for cell wall material (Le Bourvellec, C. “Non-covalent interaction between procyanidins and apple cell wall material: Part I. Effect of some environmental parameters.” Biochim. Biophys. Acta. 2004, 1672, 192-202), with higher molecular weight compounds having a greater affinity for binding than smaller compounds.
The idea of “unextractable” procyanidins has been of great interest because it is believed that the procyanidin contents in plant materials has been underestimated due to the presence of procyanidins bound so tightly to cell wall material that they are not released by conventional extraction methods. (Hellström, J. K., et al. “HPLC determination of extractable and unextractable proanthocyanidins in plant materials.” J. Agric. Food Chem. 2008, 56, 7617-7624; Madhujith, T., et al. “Antioxidant potential of barley as affected by alkaline hydrolysis and release of insoluble-bound phenolics.” Food Chem. 2009, 117, 615-620; Hellström, J. K., et al. “Proanthocyanidins in common food products of plant origin.” J. Agric. Food Chem. 2009, 57, 7899-7906; Arranz, S., et al. “High contents of nonextractable polyphenols in fruits suggest that polyphenol contents of plant foods have been underestimated.” J. Agric. Food Chem. 2009, 57, 7298-7303; and Perez-Jimenez, J., et al. “Proanthocyanidin content in foods is largely underestimated in the literature data: An approach to quantification of the missing proanthocyanidins.” Food Res. Int. 2009, 42, 1381-1388). Hellström, et al. (“HPLC determination of extractable and unextractable proanthocyanidins in plant materials.” J. Agric. Food Chem. 2008, 56, 7617-7624) determined unextractable procyanidins in plant materials by acid-catalyzed depolymerization of the compounds into flavan-3-ols and benzylthioethers using thioacidolysis and determined the amount of unextractable procyanidins in several plant materials including cranberries. (Hellström, J. K., et al. “Proanthocyanidins in common food products of plant origin.” J. Agric. Food Chem. 2009, 57, 7899-7906). Other researchers have used butanol:HCl with heat to determine the amount of bound procyanidins. (Arranz, S., et al. “High contents of nonextractable polyphenols in fruits suggest that polyphenol contents of plant foods have been underestimated.” J. Agric. Food Chem. 2009, 57, 7298-7303; and Ossipova, S., et al. “Proanthocyanidins of mountain birch leaves: quantification and properties.” Phytochem. Anal. 2001, 12, 128-133). The later is based on the principle that under heat and acid, procyanidins are converted to cyanidin, which can be measured spectrophotometrically. Researchers found that apples, peaches and nectarines contain higher levels of non-extractable procyanidins than extractable procyanidins. (Arranz, S., et al. “High contents of nonextractable polyphenols in fruits suggest that polyphenol contents of plant foods have been underestimated.” J. Agric. Food Chem. 2009, 57, 7298-7303).
The foregoing conventional extraction methods are effective in identifying the presence of bound procyanidins; however, problems exist when using these methods for quantification because of the kinetics of the reactions. Thiolysis yields have been reported to be low (34-63%), and this may be due to impurities, thiolysis resistant bonds or instability of reaction products. (Matthews, S., et al. “Method for estimation of proanthocyanidins based on their acid depolymerization in the presence of nucleophiles.” J. Agric. Food Chem. 1997, 45, 1195-1201). The butanol:HCl assay produces several side reactions, which result in lower yields, and not all procyanidins react the same under the reaction conditions. (Hummer, W., et al. “Analysis of proanthocyanidins.” Mol. Nutr. Food Res. 2008, 52, 1381-1398). Additionally, these methods do not preserve the integrity of the procyanidins; therefore, they are unrecoverable. These methods generally use acid and heat to release the procyanidins, and under these conditions, the procyanidins are depolymerized and converted to anthocyanins, such as cyanidin. Cyanidin is an unstable compound and does not have the same biological function as procyanidins, and therefore, while these methods may be an effective means of estimating the amount of bound procyanidins, they are unrecoverable due to the reaction conditions.
Alkaline treatments have been used to extract bound phenolic acids and other phenolic compounds from cereal grains and grasses, such as rice, wheat and corn. The phenolic compounds, namely ferulic acid and p-coumaric acid, are insoluble and bound to cell wall materials where many of the compounds are esterified to cell wall polysaccharides or bound to lignin with ether linkages. Treatment with different concentrations of sodium hydroxide for varying lengths of time has proven to be effective in releasing these bound phenolic compounds from grains. (Adom, K. K., et al. “Antioxidant activity of grains.” J. Agric. Food Chem. 2002, 50, 6182-6187; and Barberousse, H., et al. “Analytical methodologies for quantification of ferulic acid and its oligomers.” J. Sci. Food Agric. 2008, 88, 1494-1511). Alkaline hydrolysis has also been used to effectively extract bound ferulic, p-coumaric, caffeic and sinapic acids from citrus peel and seeds. (Bocco, A., et al. “Antioxidant activity and phenolic composition of citrus peel and seed extracts.” J. Agric. Food Chem. 1998, 46, 2123-2129). In fruits, however, conventional extraction methods do not utilize an alkaline treatment to release bound phenolic compounds because many phenolic compounds in fruits, including anthocyanins, are unstable in alkaline conditions. Additionally, harsh processing conditions such as alkaline hydrolysis may result in depolymerization of polymeric procyanidins to release lower molecular weight procyanidins such as monomers and dimers. This phenomenon may be difficult to distinguish from enhanced extraction, but may play an important role in the apparent increased extraction of procyanidins.
Cranberries (Vaccinium macrocarpon) are growing in popularity due to the increasing information regarding their health benefits. Cranberry juice has long been recognized for its ability to prevent urinary tract infections. In addition, there are several other health benefits associated with cranberries, including antioxidant, antitumor, antiulcer, anti-inflammatory and antiatherosclerotic activities. (Reed, J. “Cranberry flavonoids, atherosclerosis and cardiovascular health.” Crit. Rev. Food Sci. Nutr. 2002, 42, 301-316; Neto, C. “Cranberry and its phytochemicals: a review of in vitro anticancer studies.” J. Nutr. 2007, 137, 186S-193; Yan, X., et al. “Antioxidant activities and antitumor screening of extracts from cranberry fruit (Vaccinium macrocarpon).” J. Agric. Food Chem. 2002, 50, 5844-5849; and Sobota, A. E. “Inhibition of bacterial adherence by cranberry juice: potential use for the treatment of urinary tract infections.” J. Urol. 1984, 131, 1013-1016).
Cranberry pomace is composed primarily of seeds, skins and stems, which are leftover from the juicing and canning processes of the cranberry processing industry. (Vattem, D. A., et al. “Solid-state production of phenolic antioxidants from cranberry pomace by Rhizopus oligosporus.” Food Biotech. 2002, 16, 189-210). The seeds and skins of cranberries are a rich source of polyphenolic compounds, which have shown to be responsible for the numerous health benefits associated with the cranberries.
In addition to cranberries, many other types of plant materials are known to contain procyanidins, such as apples, pine bark, cinnamon, cocoa, grapes, bilberry, black currant, green tea, black tea, chokeberry, blueberry and sorghum.
It is therefore desirable to provide a process of using alkaline hydrolysis to extract procyanidins, which are not extracted by conventional extraction methods.
It is further desirable to provide a process of extracting procyanidins from procyanidin-containing plant materials using alkaline hydrolysis followed by acidification in order to release the procyanidins in a stable form.
It is still further desirable to provide a process of extracting bound procyanidins by alkaline hydrolysis that may be utilized to estimate the amount of bound or unextractable procyanidins in procyanidin-containing plant materials.
It is yet further desirable to provide a process of extracting procyanidins by alkaline hydrolysis that may be utilized industrially as a process of recovering procyanidins from procyanidin-containing plant materials, which may subsequently be used in dietary supplements or added to products to enhance health benefits, for example, protection against urinary tract infections.
It is yet further desirable to provide a process of extracting procyanidins by alkaline hydrolysis that enhances the bioavailability of procyanidin oligomers, namely monomeric, dimeric and trimeric procyanidins.
It is yet further desirable to provide a process of extracting procyanidins by alkaline hydrolysis capable of separating low molecular weight procyanidins (monomers, dimers and trimers) from high molecular weight procyanidins.
It is still yet further desirable to provide a process of extracting procyanidins by alkaline hydrolysis that may be utilized on a resulting residue from conventional extraction methods in order to increase the procyanidin extraction yield.